Liquid preparation of anti-TNFR2 antibody as well as preparation method and application of liquid preparation

Abstract

The invention discloses a liquid preparation of an anti-TNFR2 (Tumor Necrosis Factor Receptor 2) antibody as well as a preparation method and application of the liquid preparation. The liquid preparation comprises (i) the anti-TNFR2 antibody and (ii) a solvent, the anti-TNFR2 antibody comprises a light chain variable region VL and a heavy chain variable region VH, and according to Kabat numbering, the VL comprises CDR1 with an amino acid sequence as shown in SEQ ID NO: 1, CDR2 with an amino acid sequence as shown in SEQ ID NO: 2 and CDR3 with an amino acid sequence as shown in SEQ ID NO: 3; the VH comprises a CDR1 of which the amino acid sequence is as shown in SEQ ID NO: 4, a CDR2 of which the amino acid sequence is as shown in SEQ ID NO: 5 and a CDR3 of which the amino acid sequence is as shown in SEQ ID NO: 6. The liquid preparation of the anti-TNFR2 antibody is good in stability and can be stably stored for 4 weeks under the severe condition of 40 DEG C, an SEC purity detection result shows that the purity of the anti-TNFR2 antibody in the liquid preparation is high, and the liquid preparation of the anti-TNFR2 antibody can guarantee the storage and medication stability of the anti-TNFR2 antibody.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and specifically relates to a liquid preparation of an anti-TNFR2 antibody and a preparation method and application thereof. Background Art

[0002] Tumor necrosis factor (TNF) is a pleiotropic cytokine with pro-inflammatory and anti-inflammatory functions. TNFRSF1B (TNFreceptor superfamily member 1b) is a member of the TNF receptor superfamily, also known as TNFR2. Its ligand TNF mainly recognizes TNFR1 for apoptosis and relies on TNFR2 for functions related to T cell survival - promoting the transcription of survival genes through NF-κB (kappa-light chain enhancer of nuclear factor activated B cells) signals, thereby promoting cell proliferation and cell survival. TNFR2 is selectively expressed as a surface oncogene in many types of tumors and has the function of promoting tumor cell proliferation. At the same time, it is also expressed in suppressive immune cells in the tumor microenvironment, including regulatory T cells (Tregs) and myeloid-derived suppressor cells, and is a potential driving force for immune escape, tumor growth, and checkpoint blockade resistance. In tumors such as acute myeloid leukemia, lung cancer, and ovarian cancer, abnormally elevated TNFR2+Treg subpopulations can participate in the formation of an immunosuppressive tumor microenvironment, weaken the body's anti-tumor immune response, assist tumor immune escape, and are associated with poor prognosis. Based on the current research on this target, TNFR2 is expected to become an ideal tumor treatment target with broad prospects for clinical treatment. Anti-TNFR2 antibodies and / or their combination therapy for tumors are expected to become new tumor treatment options, bringing new possibilities for tumor patients. However, there is a lack of anti-TNFR2 antibody-related preparations on the market, and there is an urgent need for an anti-TNFR2 antibody preparation with good stability, structure and function. Summary of the invention

[0003] In order to solve the above technical problems, the present invention provides a liquid preparation of an anti-TNFR2 antibody, a preparation method and an application thereof. The anti-TNFR2 monoclonal antibody of the present invention (hereinafter referred to as monoclonal antibody A) can specifically bind to TNFR2, thereby regulating the tumor microenvironment and killing tumor cells. The liquid preparation of the anti-TNFR2 antibody of the present invention has good stability, and can be stably stored for 4 weeks under severe conditions of 40°C, and the SEC purity test results show that the purity of the antibody liquid preparation of the present invention is relatively high. The monoclonal antibody A of the present invention provides a guarantee for the structural and functional stability during the production, transportation and storage of antibodies.

[0004] The first aspect of the present invention provides a liquid preparation of an antibody, comprising:

[0005] (i) an anti-TNFR2 antibody, and

[0006] (ii) solvent;

[0007] Wherein, the anti-TNFR2 antibody comprises a light chain variable region VL and a heavy chain variable region VH. According to Kabat numbering, the VL comprises a CDR1 as shown in SEQ ID NO: 1, a CDR2 as shown in SEQ ID NO: 2, and a CDR3 as shown in SEQ ID NO: 3; the VH comprises a CDR1 as shown in SEQ ID NO: 4, a CDR2 as shown in SEQ ID NO: 5, and a CDR3 as shown in SEQ ID NO: 6.

[0008] The solvent described in the present invention may be conventional in the art, for example, a pharmaceutically acceptable solvent, such as a pharmaceutically acceptable solvent for antibody preparations.

[0009] In a preferred embodiment, in the liquid preparation, the VL of the anti-TNFR2 antibody comprises the amino acid sequence shown in SEQ ID NO:7, and the VH comprises the amino acid sequence shown in SEQ ID NO:8.

[0010] In a preferred embodiment, the anti-TNFR2 antibody comprises a light chain having an amino acid sequence as shown in SEQ ID NO:9, and a heavy chain having an amino acid sequence as shown in SEQ ID NO:10, and the antibody is preferably a full-length antibody, Fab, Fab', F(ab')2, Fv such as scFv or di-scFv, VHH or heavy chain antibody (HCAb).

[0011] The concentration of the anti-TNFR2 antibody of the present invention can be conventional in the art, especially an acceptable concentration value in antibody preparations.

[0012] In a preferred embodiment, the concentration of the anti-TNFR2 antibody is 1-200 mg / ml, preferably 5-150 mg / ml, and more preferably 10-100 mg / ml.

[0013] In a preferred embodiment, the concentration of the anti-TNFR2 antibody is 10 mg / ml, 20 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, 80 mg / ml, 100 mg / ml, 120 mg / ml, 130 mg / ml, 150 mg / ml or 180 mg / ml.

[0014] In a preferred embodiment, the solvent is:

[0015] (a) water such as ultrapure water; or, (b) a buffer solution having a pH of 4.0 to 7.0.

[0016] In a preferred embodiment, the pH of the buffer is 4.5, 5.0, 5.5, 6.0 or 6.5.

[0017] In a preferred embodiment, (a) further contains a surfactant, preferably a polysorbate surfactant.

[0018] In a preferred embodiment, (a) contains 0.1-2 mg / ml of polysorbate 80, such as 0.1 mg / ml of polysorbate 80.

[0019] In a preferred embodiment, in (b), the buffer comprises one or more of an acetate buffer system, a phosphate buffer system, a histidine hydrochloride buffer and a histidine acetate buffer.

[0020] In a preferred embodiment, the buffer is an acetate buffer at pH 4.5 to 5.8, a histidine hydrochloride buffer at pH 5.0 to 6.5, a histidine acetate buffer at pH 4.5 to 6.5, or a phosphate buffer at pH 6.0 to 7.0.

[0021] In a preferred embodiment, the concentration of the buffer is 5-50 mM, preferably 5-30 mM.

[0022] In a preferred embodiment, the concentration of the buffer is 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM or 45 mM.

[0023] In a preferred embodiment, the liquid preparation further comprises: (iii) a stabilizer.

[0024] Preferably, the stabilizer comprises an osmotic pressure regulator and / or an antioxidant: the osmotic pressure regulator is one or more of sodium chloride, sucrose, trehalose, sorbitol, arginine, glycine and proline; and / or the antioxidant is selected from one or more of glycine, methionine, arginine hydrochloride, sodium chloride and disodium edetate.

[0025] Among them, arginine, glycine and proline are types of osmotic pressure regulators that reduce viscosity.

[0026] Further preferably, the concentration of the osmotic pressure regulator is 1 to 300 mM, preferably 100 to 300 mM, and more preferably 147 to 273 mM.

[0027] In a preferred embodiment, the concentration of the osmotic pressure regulator is 10 mM, 50 mM, 100 mM, 150 mM, 200 mM or 250 mM.

[0028] Further preferably, the concentration of the antioxidant is 1 to 50 mM, preferably 1 to 20 mM, more preferably 7 to 13 mM.

[0029] In a preferred embodiment, the concentration of the antioxidant is 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM or 45 mM.

[0030] In a preferred embodiment, the liquid preparation further comprises: (iv) a surfactant.

[0031] Preferably, the surfactant is poloxamer 188 or a polysorbate surfactant, such as polysorbate 80.

[0032] In a preferred embodiment, the concentration of the surfactant is 1 to 2 mg / ml, preferably 0.05 to 0.5 mg / ml, and more preferably 0.1 to 0.15 mg / ml.

[0033] In a preferred embodiment, the liquid preparation further comprises: (v) a chelating agent, such as edetate disodium.

[0034] In a preferred embodiment, the concentration of the chelating agent is 0.01-0.1 mg / mL, preferably 0.01-0.05 mg / mL, for example 0.019 mg / mL.

[0035] In a preferred embodiment, the liquid preparation is an injection, preferably for subcutaneous injection or intravenous injection, or an infusion, such as for intravenous infusion.

[0036] In a preferred embodiment, the liquid preparation comprises:

[0037] (i) 10 to 30 mg / ml of anti-TNFR2 antibody;

[0038] (ii) a stabilizer comprising 147-273 mM sucrose and 7-13 mM methionine

[0039] (iii) 0.1 to 0.15 mg / ml polysorbate 80;

[0040] (iv) 5-30 mM acetate buffer system, pH 4.5-6.0.

[0041] In a specific embodiment, the liquid formulation comprises:

[0042] (1) 5 mM acetate buffer system, pH 5.0, 147 mM sucrose, 7 mM methionine, 0.05 mg / ml polysorbate 80 and 10 mg / ml anti-TNFR2 antibody; or,

[0043] (2) 10 mM acetate buffer system, pH 5.0, 210 mM sucrose, 10 mM methionine, 0.10 mg / ml polysorbate 80, and 20 mg / ml anti-TNFR2 antibody; or,

[0044] (3) 10 mM acetate buffer system, pH 5.4, 147 mM sucrose, 10 mM methionine, 0.10 mg / ml polysorbate 80, and 20 mg / ml anti-TNFR2 antibody; or,

[0045] (4) 10 mM acetate buffer system, pH 5.4, 273 mM sucrose, 10 mM methionine, 0.10 mg / ml polysorbate 80, and 20 mg / ml anti-TNFR2 antibody; or,

[0046] (5) 10 mM acetate buffer system, pH 5.4, 210 mM sucrose, 10 mM methionine, 0.10 mg / ml polysorbate 80, and 20 mg / ml anti-TNFR2 antibody; or,

[0047] (6) 10 mM acetate buffer system, pH 5.4, 210 mM sucrose, 7 mM methionine, 0.10 mg / ml polysorbate 80, and 20 mg / ml anti-TNFR2 antibody; or,

[0048] (7) 10 mM acetate buffer system, pH 5.4, 210 mM sucrose, 13 mM methionine, 0.10 mg / ml polysorbate 80, and 20 mg / ml anti-TNFR2 antibody; or,

[0049] (8) 5 mM acetate buffer system, pH 5.4, 210 mM sucrose, 10 mM methionine, 0.10 mg / ml polysorbate 80 and 20 mg / ml anti-TNFR2 antibody; or,

[0050] (9) 15 mM acetate buffer system, pH 5.4, 210 mM sucrose, 10 mM methionine, 0.10 mg / ml polysorbate 80, and 20 mg / ml anti-TNFR2 antibody; or,

[0051] (10) 15 mM acetate buffer system, pH 6.0, 273 mM sucrose, 13 mM methionine, 0.15 mg / ml polysorbate 80, and 30 mg / ml anti-TNFR2 antibody; or,

[0052] (11) 10 mM acetate buffer system, pH 6.0, 210 mM sucrose, 10 mM methionine, 0.10 mg / ml polysorbate 80 and 20 mg / ml anti-TNFR2 antibody; or,

[0053] (12) 10 mM acetate buffer system, pH 5.4, 210 mM sucrose, 10 mM methionine, 0.05 mg / ml polysorbate 80, and 20 mg / ml anti-TNFR2 antibody; or,

[0054] (13) 10 mM acetate buffer system, pH 5.4, 210 mM sucrose, 10 mM methionine, 0.15 mg / ml polysorbate 80 and 20 mg / ml anti-TNFR2 antibody; or,

[0055] (14) 10 mM acetate buffer system, pH 5.4, 210 mM sucrose, 10 mM methionine, 0.10 mg / ml polysorbate 80 and 10 mg / ml anti-TNFR2 antibody; or,

[0056] (15) 10 mM acetate buffer system, pH 5.4, 210 mM sucrose, 10 mM methionine, 0.10 mg / ml polysorbate 80 and 30 mg / ml anti-TNFR2 antibody; or,

[0057] (16) 20 mM, pH 5.4 acetate buffer system and 7.8 mg / ml anti-TNFR2 antibody; or,

[0058] (17) 20 mM, pH 6.0 phosphate buffer system and 7.8 mg / ml anti-TNFR2 antibody; or,

[0059] (18) ultrapure water, 0.1 mg / ml polysorbate 80, and 7.8 mg / ml anti-TNFR2 antibody; or,

[0060] (19) ultrapure water and 7.8 mg / ml anti-TNFR2 antibody; or,

[0061] (20) 20 mM, pH 6.0 phosphate buffer system, 220 mM sucrose, 0.20 mg / ml polysorbate 80, and 20 mg / ml anti-TNFR2 antibody; or,

[0062] (21) 20 mM, pH 6.0 phosphate buffer system, 220 mM trehalose, 0.20 mg / ml polysorbate 80 and 20 mg / ml anti-TNFR2 antibody; or,

[0063] (22) 20 mM, pH 6.0 phosphate buffer system, 210 mM sucrose, 10 nM methionine, 0.20 mg / ml polysorbate 80 and 20 mg / ml anti-TNFR2 antibody; or,

[0064] (23) 20 mM acetate buffer system, pH 5.4, 210 mM sucrose, 10 nM methionine, 0.20 mg / ml polysorbate 80, and 20 mg / ml anti-TNFR2 antibody.

[0065] The second aspect of the present invention provides a solid antibody preparation obtained by solidifying the liquid preparation as described in the first aspect of the present invention.

[0066] Preferably, the solid antibody preparation is in the form of a lyophilized powder injection.

[0067] The third aspect of the present invention provides a method of adding an anti-TNFR2 antibody to a solvent to form the liquid preparation as described in the first aspect of the present invention.

[0068] In a preferred embodiment, the solvent is as defined in the liquid preparation described in the first aspect of the present invention.

[0069] In a preferred embodiment, the solvent further contains a stabilizer, a surfactant and / or a chelating agent as defined in the liquid preparation described in the first aspect of the present invention.

[0070] The fourth aspect of the present invention provides a delivery device comprising the liquid preparation according to the first aspect of the present invention or the solid antibody preparation according to the second aspect of the present invention.

[0071] The fifth aspect of the present invention provides a prefilled syringe comprising the liquid preparation according to the first aspect of the present invention or the solid antibody preparation according to the second aspect of the present invention, for intravenous injection or intramuscular injection.

[0072] The sixth aspect of the present invention provides a use of the liquid preparation according to the first aspect of the present invention or the solid antibody preparation according to the second aspect of the present invention in the preparation of diagnosis, prevention and / or treatment of cancer.

[0073] Preferably, the cancer is ovarian cancer, acute myeloid leukemia, Lewis lung cancer, breast cancer, hepatocellular carcinoma, neural cancer and / or colorectal cancer, preferably colon cancer, such as colon adenocarcinoma.

[0074] Unless otherwise specified, the concentrations described in the present invention are the working concentrations of the substances.

[0075] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0076] The reagents and raw materials used in the present invention are commercially available.

[0077] The positive and progressive effects of the present invention are:

[0078] The liquid preparation of the anti-TNFR2 antibody of the present invention has good stability, and can be stably stored for 4 weeks under the severe condition of 40°C, and the SEC purity test results show that the purity of the anti-TNFR2 antibody in the liquid preparation of the present invention is relatively high (up to 95.0% or more). Therefore, the liquid preparation of the anti-TNFR2 antibody of the present invention can ensure the storage and medication stability of the anti-TNFR2 antibody, and has broad industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 It is a drug concentration-time curve showing the change of antibody drug concentration in the serum of humanized TNFR2 mice after injection of mAb A. DETAILED DESCRIPTION

[0080] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0081] Part I

[0082] This part includes the preparation, pharmacodynamics test and in vivo efficacy test of the anti-TNFR2 antibody of the present invention (hereinafter referred to as mAb A).

[0083] Example 1: Preparation of the Antibody of the Invention

[0084] To generate antibodies against human TNFR2 (hTNFR2; SEQ ID NO: 18), RenMab mice were immunized with human TNFR2. mAb A was prepared by the method described below.

[0085] The construction method of RenMab mice is as follows: 1) modifying the human immunoglobulin region on the human chromosome; 2) introducing the modified human chromosome into the mouse recipient cell; 3) replacing the mouse immunoglobulin variable region with the human immunoglobulin variable region by direct replacement (e.g., homologous recombination or Cre-mediated recombination). In some cases, the human immunoglobulin variable region can be introduced into the mouse genome by a step-by-step method. Then, the correctly replaced recipient cells are screened. The cells are then injected into blastocysts to prepare chimeric mice. Finally, breeding is performed to obtain mice containing complete human immunoglobulin variable regions (a detailed description of the construction of RenMab mice can be found in patent document CN112400022A).

[0086] Mouse immunization

[0087] RenMab mice were immunized with Fc-tagged human TNFR2 protein. The Fc-tagged human TNFR2 protein was emulsified with adjuvant and injected into four locations on the back of the mouse. For the first subcutaneous injection (sc), the diluted antigen was emulsified with an equal volume of complete Freund's adjuvant (CFA). In subsequent subcutaneous injections, the protein was emulsified with an equal volume of incomplete Freund's adjuvant (IFA). At least four injections were performed, with at least 14 days between each two injections. Seven days after the third injection or booster immunization, blood (serum) was collected and analyzed for antibody titer using fluorescence activated cell sorting (FACS).

[0088] In another experiment, mice were immunized by injecting an expression plasmid encoding human TNFR2 into mice. The plasmid encoding the antigen was injected into the tibialis anterior muscle (intramuscular injection; im). At least four injections were performed, with at least 14 days between each injection. Blood (serum) was collected seven days after the last immunization, and the serum was tested for antibody titer by ELISA.

[0089] In addition, a booster immunization procedure was performed at least fourteen days after immunization (by plasmid injection or protein injection). CHO cells (purchased from Thermo Fisher Scientific) expressing TNFR2 antigen on the surface were injected into the mouse tail vein via the tail vein. Immune system organs (e.g., bone marrow, lymph nodes, spleen, etc.) were then collected four days after the injection.

[0090] Spleen cells were collected and fused with mouse myeloma cells to maintain their viability and form hybridoma cell lines. Hybridoma cells were screened and selected to identify cell lines that produced monoclonal antibody A. Using this technology and the above-mentioned immunogen, several anti-TNFR2 chimeric antibodies (i.e., antibodies with human variable domains and mouse constant domains) were obtained. Specifically, after pulse immunization, mouse immune organs were harvested and plasma cells were separated by magnetic beads. Monoclonal hybridoma cells that secrete antigen-specific monoclonal antibodies were screened using hybridoma fusion technology. Antibody light chain and heavy chain variable (V) region sequences were obtained by reverse transcription and PCR sequencing in selected monoclonal hybridoma cells. The antibody light chain and heavy chain V region sequences were constructed into antibody expression vectors and verified by the Expi CHO-STM cell expression system. Cells were transfected in a 24-well system and antibodies were collected in the supernatant on the 3rd day. FACS was used to verify the specificity of binding between the antibody and TNFR2. The constant domains in the obtained anti-TNFR2 chimeric antibodies can be easily replaced to obtain fully human anti-TNFR2 antibodies (anti-hTNFR2 antibodies).

[0091] Antigen-positive B cells were also isolated directly from immunized mice without fusion with myeloma cells. Anti-TNFR2 antibodies were further isolated from antigen-positive B cells. The light chain and heavy chain V region sequences of the antibodies were obtained directly from antigen-positive B cells. For example, single cell techniques (e.g., Optofluidic system, Digital Cell Biology Company (BerkeleyLights Inc.) is used to screen and discover plasma cells that secrete antigen-specific monoclonal antibodies. Antibody V region sequences expressing antibodies are obtained using reverse transcription and PCR sequencing. FACS is used to verify the binding between antibodies and TNFR2. By this method, when these antibodies are connected with IgG1, several anti-TNFR2 antibodies can be obtained, and after screening, monoclonal antibody A of the present invention is obtained.

[0092] According to the Kabat numbering definition, the CDR sequence of monoclonal antibody A includes the sequences of CDR1-CDR3 of the light chain variable domain as shown in SEQ ID NO: 1-3, and the sequences of CDR1-CDR3 of the heavy chain variable domain as shown in SEQ ID NO: 4-6. CDR can also be defined by the IMGT system. According to the IMGT numbering, the sequence of CDR2 of the light chain variable domain is SAS, the sequences of CDR1 and CDR3 are shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively, and the sequences of CDR1-CDR3 of the heavy chain variable domain are shown in SEQ ID NO: 13-15, respectively.

[0093] Kabat Number:

[0094] Light chain CDR1: RASQSVSSNLA (SEQ ID NO: 1)

[0095] Light chain CDR2: SASTRAT (SEQ ID NO: 2)

[0096] Light chain CDR3: QQYSNWPFT (SEQ ID NO: 3)

[0097] Heavy chain CDR1: IYGMH (SEQ ID NO: 4)

[0098] Heavy chain CDR2: LISYDGNDKYFADSVKG (SEQ ID NO: 5)

[0099] Heavy chain CDR3: AVDTAVVTFFDY (SEQ ID NO: 6)

[0100] IMGT Number:

[0101] Light chain CDR1: QSVSSN (SEQ ID NO: 11)

[0102] Light chain CDR2: SAS

[0103] Light chain CDR3: QQYSNWPFT (SEQ ID NO:12)

[0104] Heavy chain CDR1: GFTFNIYG (SEQ ID NO:13)

[0105] Heavy chain CDR2: ISYDGNDK (SEQ ID NO:14)

[0106] Heavy chain CDR3: AKAVDTAVVTFFDY (SEQ ID NO:15)

[0107] Light chain variable region (SEQ ID NO:7):

[0108] EIVMTQSPVTLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYSASTRAT GIPARFSGSGSGTEFTLTISSLQSEDLAVYYCQQYSNWPFTFGPGTKVDIK

[0109] Heavy chain variable region (SEQ ID NO:8):

[0110] QVQLVESGGGVVHPGKSLRLSCAASGFTFNIYGMHWVRQAPGKGLEWVALISYDGNDKYFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAVDTAVVTFFDYWGQGTLVTVSS

[0111] Light chain (SEQ ID NO:9):

[0112] EIVMTQSPVTLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYSASTRATGIPARFSGSGSGTEFTLTISSLQSEDLAVYYCQQYSNWPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0113] Heavy chain (SEQ ID NO:10):

[0114] QVQLVESGGGVVHPGKSLRLSCAASGFTFNIYGMHWVRQAPGKGLEWVALISYDGNDKYFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAVDTAVVTFFDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0115] Antibody preparation

[0116] The positive antibody sequences in the sequence verification stage were subjected to plasmid extraction and transfection into a 25 mL system. After 10-12 days of cell culture, the expression supernatant was collected and affinity chromatography was performed. The obtained antibody samples were used for the following in vitro tests and screening.

[0117] Example 2. In vitro testing of mAb A

[0118] Cross-reactivity of mAb A to mouse, dog, and monkey TNFR2

[0119] In each experiment, CHO cells were transfected with EGFP and human TNFR2 (TNFR2, SEQ ID NO: 18), mouse TNFR2 (mTNFR2, SEQ ID NO: 17), monkey (Crab-eating monkey) TNFR2 (fTNFR2, SEQ ID NO: 19), or dog (canine) TNFR2 (dTNFR2, SEQ ID NO: 20).

[0120] 30 μl CHO cells (1×10 5 30 μl of purified mAb A (10 μg / ml) (as listed in Table 1) was added to each well and incubated at 4°C for 30 minutes.

[0121] After washing twice with PBS (1600 rpm, 6 min), 50 μl of Alexa Fluor-labeled mAb A was added to each well at a dilution of 1:500, incubated at 4°C for 15 min, and then washed with PBS (1200 rpm, 5 min). The signal of AF647 was detected by flow cytometry.

[0122] The following table shows the cross-reactivity of mAb A with human (TNFR2), mouse (mTNFR2), monkey (fTNFR2) and dog (dTNFR2) TNFR2.

[0123] Table 1

[0124] TNFR2 (human) mTNFR2 (mouse) fTNFR2 (monkey) dTNFR2 (dog) MAb A have none have none

[0125] In vitro ADCC assay

[0126] Experiments were conducted to evaluate the ADCC effect of the monoclonal antibody A of the present invention. In the experiments, isotype control human IgG1 (Crown Bioscience Inc., C0001-3) was used.

[0127] Lactate dehydrogenase (LDH) is a cytoplasmic enzyme found in many different cell types and is released into the cell culture medium upon plasma membrane damage. The CyQUANT LDH Cytotoxicity Assay Kit (Invitrogen, catalog number: C20301) was used to accurately quantify extracellular LDH to assess the ADCC effect of antibodies.

[0128] The monoclonal antibody A was serially diluted (10 times) to a maximum concentration of 100 μg / mL. The target cells (MC38 cells overexpressing human TNFR2 (purchased from Shunran (Shanghai) Biotechnology Co., Ltd.)) were seeded in a 96-well plate (cell density was 2×10 4 cells / well, 100 μL) and incubated at 37°C for 3-4 hours. Effector cells (peripheral blood mononuclear cells (PBMC)) were revived (cell density was 2×10 4 cells / well). The same volume (100 μL) of effector cells was added to each well of a 96-well plate together with 10 μL of antibody. The above 96-well plate was incubated overnight at 37°C. The absorbance values ​​at 490 nm and 680 nm were detected by an ELISA reader and used to calculate the killing of target cells (% cytotoxicity) by each group of antibodies. EC was calculated using antibody concentration as the horizontal axis and cytotoxicity as the vertical axis using nonlinear fitting. 50 value.

[0129]

[0130] In the above formula, "experimental" refers to the absorbance value of the experimental well; "effector" refers to the absorbance value of the effector cell. "Target" refers to the absorbance value of the target cell. "Spontaneous" refers to the autofluorescence absorbance value of the cell culture medium. "Target maximum" refers to the highest absorbance value of the target cell. "Spontaneous" refers to the absorbance value of the target cell. * ” refers to the absorbance value of the cell culture medium volume control well.

[0131] EC 50 The results are shown in Table 2. Compared with the isotype control hIgG1, the killing of target cells (% cytotoxicity) by the mAb A of the present invention increased with increasing doses of these antibodies, indicating that the mAb A of the present invention has ADCC activity.

[0132] Table 2

[0133]

[0134] In another similar experiment, target cells (MC38 cells overexpressing human TNFR2) and effector cells (FcR-TANK (CD16a-158V) cell line, Immune Onco Biopharmaceuticals (Shanghai) Co., Ltd.) were used to evaluate the ADCC effect of mAb A.

[0135] EC 50 The results are shown in Table 2. Compared with the isotype control hIgG1, the killing of target cells (% cytotoxicity) by mAb A increased with increasing doses of these antibodies, indicating that it has a stronger ADCC activity.

[0136] Table 2

[0137] protein ADCC <![CDATA[EC 50 ng / mL]]> MAb A have 80.69 hIgG1 none —

[0138] Example 3. In vivo testing of mAb A

[0139] In order to test the antibody in vivo and predict the effect of mAb A in humans, a humanized TNFR2 mouse model was generated. The humanized TNFR2 mouse model was engineered to express a chimeric TNFR2 protein (SEQ ID NO: 16), wherein the extracellular region of the mouse TNFR2 protein was replaced by the corresponding extracellular region of human TNFR2. Amino acid residues 33-260 of mouse TNFR2 (SEQ ID NO: 17) were replaced by amino acid residues 33-259 of human TNFR2 (SEQ ID NO: 18).

[0140] Humanized TNFR2 mouse models (e.g., B-TNFR2 mice) provide a new tool for testing new therapeutic treatments in a clinical setting by significantly reducing the difference between the clinical outcomes of humans and ordinary mice expressing mouse TNFR2. A detailed description of the humanized TNFR2 mouse model can be found in PCT / CN2020 / 113618; it is incorporated herein by reference in its entirety.

[0141] mAb A was tested in TNFR2 humanized mice (B-TNFR2) to demonstrate its effect on tumor growth in vivo.

[0142] In vivo efficacy in B-TNFR2 mice with MC38, 10 mg / kg

[0143] MC-38 tumor cells (colon adenocarcinoma cells) were injected subcutaneously into B-TNFR2 mice. When the tumor volume of the mice reached 100 mm 3 -150 mm 3 At the same time, the mice were randomly divided into different groups (six mice in each group) according to the volume of the tumor.

[0144] The mice were then injected with PBS as a control (G1), monoclonal antibody A, and anti-mPD-1 (G9) (BIO X CELL, catalog number: BE0146). Anti-mPD-1, which has been shown to be effective in mice, was used as a positive control. The antibody was administered by intraperitoneal injection at 10 mg / kg on the first and fourth days of each week for 3 weeks (6 injections in total).

[0145] The weight of mice was monitored throughout the treatment period. The weight of mice in different groups increased, with no statistically significant differences (P>0.05). On the day of grouping (day 0; "D0"), the average body weight of each group was in the range of 20.3g-21.4g. At the end of the experiment (28 days after grouping, D28), the average body weight of each group was in the range of 22.7g-24.7g, and the weight change was in the range of 107.4%-116.5%. The results show that mAb A is well tolerated and non-toxic to mice.

[0146] The tumor size showed significant differences in the groups treated with mAb A: TGI of mAb A on day 28 (28 days after grouping) TV The positive control (anti-mPD-1, G9) showed good therapeutic effect (TGI TV %=78.5%) mAb A showed good therapeutic effect (TGI TV %>60%).

[0147] Table 3

[0148]

[0149] Example 4. PK and TILS analysis

[0150] PK analysis of B-TNFR2 mice with MC38 model at 10 mg / kg

[0151] The pharmacokinetic clearance of mAb A was determined in humanized TNFR2 mice. MC38 cells (5×10 5 ) was injected subcutaneously into humanized TNFR2 mice and the tumors were grown to 300 mm 3 The mice were divided into 8 groups (n=4) at 1:10 pm. 10 mg / kg of mAb A or isotype control IgG1 was administered by intravenous injection. Blood samples were collected at 15 min, 6 hours, 24 hours, 3 days, 5 days and 7 days after administration.

[0152] Serum levels of human antibodies were measured by sandwich ELISA enzyme-linked immunosorbent assay. In brief, goat polyclonal anti-human IgG (Fc specific) capture antibody (Jackson ImmunoResearch, catalog number: 109-036-098) was diluted to a final concentration of 2.0 μg / mL with 0.1 M carbonate buffer (pH 9.6), added to a 96-well plate (ELISA plate) at 100 μL / well, and incubated overnight at 4 ° C. Then, 200 μL of blocking buffer (2% BSA) was added to each well. The wells were sealed and incubated at room temperature for 1 hour. After washing the plate with a plate washer, horseradish peroxidase (HRP)-conjugated goat polyclonal anti-human IgG (Fc specific) antibody (Jackson ImmunoResearch, catalog number: 109-005-088) was added to each well of the ELISA plate at 100 μL / well and incubated at 37 ° C for 1 hour. After washing the plate, tetramethylbenzidine (TMB) solution was added to 96-well plates at 100 μL / well to react with HRP as a substrate. After incubation at room temperature in the dark, 100 μL stop solution (Beyotime, catalog number: P0215) was added to each well. The absorbance value of each well at 450nm and 630nm wavelength was read using an ELISA reader. Data analysis software Gen5 was used to analyze the data. The absorbance value and corresponding concentration of the calibration sample prepared by each test product were used to create a standard curve of four parameters. The antibody concentration of each serum sample was calculated using the standard curve. The drug concentration-time curve was created using the sample concentration calculated at each time point. Phoenix winnolin 8.3 was used to calculate pharmacokinetic parameters. The results are shown in Table 4 below.

[0153] Table 4

[0154]

[0155] T 1 / 2: Terminal half-life;

[0156] C max : maximum concentration;

[0157] AUC 0-7天 : Area under the blood concentration-time curve from 0 to 7 days

[0158] CL: Clearance

[0159] The above results show that after injection of monoclonal antibody A, the antibody concentration in the serum of TNFR2 humanized mice decreased over time (see Figure 1 ), which is consistent with the pharmacokinetic characteristics. The shortest half-life of mAb A in mice is 0.86 days. By the end of the seventh day of sampling, the CL of mAb A was in the range of 47.07-61.19 ml / hr / kg. The results show that the clearance efficiency of mAb A is low and its metabolism in mice is slow.

[0160] Other sequences involved in this section are shown in the following table:

[0161]

[0162]

[0163] Part 2

[0164] The following analyzes the effects of various components in the preparation on the stability of the liquid preparation (such as buffers, stabilizers, surfactants, DOE experiments of the formulation, accelerated stability and long-term stability of the formulation, etc.), and the monoclonal antibody A used is in the form of a full-length antibody. The SEC-HPLC analysis method used is as follows: a high-performance liquid chromatograph connected to a UV detector and a TSKgel G3000SWxl column are used, a phosphate and sodium sulfate mixed solution with a pH of 6.7 is used as the mobile phase, and the sample is detected at a detection wavelength of 280nm, and the concentration of the sample is calculated by the area normalization method.

[0165] Example 5 Effect of buffer on the stability of liquid preparations

[0166] The main purpose of this example is to evaluate the effect of the monoclonal antibody A of the present invention on the stability of the liquid preparation under buffers with different pH values ​​(acetate buffer, histidine hydrochloride buffer, histidine acetate buffer, phosphate buffer) and without adding a buffer.

[0167] The formulation shown in Table 5 was observed at 40±2°C for one month and evaluated by SEC-HPLC (size exclusion chromatography), wherein the concentration of monoclonal antibody A was 7.8 mg / ml.

[0168] Table 5 Buffer system and pH screening experiment screening prescription design

[0169]

[0170] Taking T0 as the initial value, the test results of different buffer systems at T0 and 40±2℃ for one month are shown in Table 6.

[0171] Table 6 Summary of SEC experimental data of different buffer systems at 40±2℃

[0172]

[0173] The test results were analyzed. From the SEC quality change trend of different buffer systems at 40°C, for monoclonal antibody A, the ultrapure water formula (A17-A18) had the best antibody stability, followed by 20mM phosphate buffer pH 6.0 and pH 6.5 (A14 and A15) and 20mM acetate buffer pH 5.4 (A3). During long-term storage, a buffer system with a suitable concentration is more conducive to maintaining protein stability. Therefore, the next step of the experiment will be mainly based on phosphate buffer, supplemented by acetate buffer and without any buffer.

[0174] Example 6 Effects of osmotic pressure regulators and antioxidants on formulation stability

[0175] The main purpose of this example is to evaluate the stability of the monoclonal antibody A of the present invention under different osmotic pressure regulators (sucrose, trehalose, sorbitol, proline) and antioxidants (glycine, methionine, arginine hydrochloride, sodium chloride, disodium edetate) conditions.

[0176] The stability of the monoclonal antibody A of the present invention was compared under the conditions of repeated freezing and thawing (-20°C (24h) to 5°C (24h) as one cycle), high temperature and horizontal shaking (200rpm) under different formulations (Table 7), wherein the O1 and O2 groups were 20mM phosphate buffer system + 220mM sucrose + 0.20mg / ml polysorbate 80. The results of SEC-HPLC (see Table 8) were used for evaluation.

[0177] Table 7 Experimental design for screening of osmotic pressure regulators and antioxidants

[0178]

[0179] Table 8 Summary of experimental data for screening of osmotic pressure regulators and antioxidants

[0180]

[0181]

[0182] Taking the data of day 0 of the group without osmotic pressure regulator (P1) as the initial value (T0), the changes of SEC-HPLC test results under different investigation conditions were investigated in different osmotic pressure regulators, different antioxidants, different surfactants and different buffer formulations.

[0183] As can be seen from Table 8, under different osmotic pressure regulator conditions, the main peak content of SEC purity in each formulation sample decreased to varying degrees under different investigation conditions. Under 40±2℃, the comprehensive stability of sucrose (P2) and trehalose (P3) formulations was better, and sucrose was preferred as the osmotic pressure regulator; under different antioxidant conditions, in repeated freeze-thaw, horizontal shaking and high temperature conditions, the SEC data of each formulation was compared, and compared with glycine (P6), arginine hydrochloride (P8), sodium chloride (P9) and disodium edetate (P10), methionine (P7) had a better antioxidant effect; under different buffer systems and different osmotic pressure regulator conditions, in repeated freeze-thaw, horizontal shaking and high temperature screening results, the SEC data of each formulation was compared, and at the same polysorbate 80 concentration, 210mM ​​sucrose + 10mM methionine (P7, A2, A3) or 250mM sucrose + 10mM methionine (W1) as osmotic pressure regulator antibodies had the best stability. Under the conditions of sucrose as an osmotic pressure regulator and methionine as an antioxidant, based on the stability test results of the monoclonal antibody A of the present invention in different buffer systems, the acetate buffer system is preferred.

[0184] Example 7 Concentration optimization experiment of formula components

[0185] The purpose of this example is to evaluate the effect of each component on the stability of the monoclonal antibody A of the present invention when it changes within a certain concentration range on the basis of determining the formulation. The experimental design is shown in Table 9, and the stability of the preparation under the conditions including but not limited to acetate buffer system (abbreviated as acetate buffer): 5-15mM; pH: 5.0-6.0; sucrose (Sucrose): 147-273mM; methionine (Met): 7-13mM; polysorbate 80: 0.05-0.15mg / ml; monoclonal antibody A concentration of the present invention: 10-30mg / ml.

[0186] Table 9 Experimental design for optimizing formulation component concentration

[0187]

[0188] The formulation shown in Table 9 was observed under illumination (at 3000Lx) for 5 days (Lx-D5), shaking (200rpm) for 3 days (ShK-D3), 5 freeze-thaw cycles (F / T5), and 40°C for one month (40°C-M1) to detect changes in the SEC purity of the monoclonal antibody A of the present invention in the preparation. The test results are shown in Table 10.

[0189] Table 10 SEC test results of formula component concentration optimization

[0190] ID T0 Lx-D5 ShK-D3 F / T5 40℃-M1 C1 99.1 99.0 99.1 99.1 95.7 C2 99.1 99.0 99.0 99.1 95.6 C3 99.1 98.9 99.0 99.1 95.5 C4 99.8 99.6 99.7 99.7 96.1 C5 99.8 99.6 99.7 99.7 96.0 C6 99.0 98.9 99.0 99.1 95.6 C7 99.8 99.6 99.7 99.7 96.1 C8 99.1 98.9 99.0 99.1 95.6 C9 99.1 98.9 99.0 99.0 95.6 C10 99.0 98.6 98.9 98.9 95.3 C11 99.0 98.8 99.0 99.0 95.5 C12 99.1 98.9 99.0 99.0 95.6 C13 99.8 99.6 99.7 99.7 96.1 C14 99.8 99.7 99.7 99.7 96.1 C15 99.1 98.8 99.0 99.0 95.5

[0191] From the results, it can be seen that under different influencing factors, the quality index change trend of the monoclonal antibody A of the present invention in the formulations with different concentration ranges is consistent, and there will be a certain degree of fluctuation compared with the target concentration, but all are within the acceptable range of the quality standard. The factor that has the greatest impact on the stability of this product is high temperature, followed by light.

[0192] Accelerated stability and long-term stability of the formulation of Example 8

[0193] The main purpose of this example is to evaluate the long-term stability of the monoclonal antibody A preparation formula D1 of the present invention at 2-8°C and the accelerated stability at 25°C.

[0194] The formulation shown in Table 11 was placed at 2-8°C and 25°C for 3 months respectively; samples were taken at each sampling point to detect the change in SEC purity of the monoclonal antibody A of the present invention in the preparation. The test results are shown in Table 12.

[0195] Table 11: Accelerated stability and long-term stability

[0196]

[0197] Table 12: Accelerated stability and long-term stability test results

[0198]

[0199] As shown in the data in Table 12, the formulation system containing 71.88 mg / mL (210 mM) sucrose, 1.49 mg / mL (10 mM) methionine, 10 mM acetate buffer and 0.1 mg / mL polysorbate 80 has a good protective effect on the monoclonal antibody A of the present invention in the formulation of the present invention. Therefore, the formulation formula shown in Table 8 can ensure good physical and chemical stability of the monoclonal antibody A of the present invention and has broad application prospects.

[0200] The above is only a preferred embodiment of the present invention. It should be noted that a person skilled in the art can make several improvements and modifications without departing from the principle of the present invention. These improvements and modifications should also be considered as the protection scope of the present invention.

Claims

1. A liquid preparation of an antibody, comprising: (i) an anti-TNFR2 antibody, and (ii) solvent; in, The anti-TNFR2 antibody comprises a light chain variable region VL and a heavy chain variable region VH. According to Kabat numbering, the VL comprises a CDR1 as shown in SEQ ID NO: 1, a CDR2 as shown in SEQ ID NO: 2, and a CDR3 as shown in SEQ ID NO: 3; the VH comprises a CDR1 as shown in SEQ ID NO: 4, a CDR2 as shown in SEQ ID NO: 5, and a CDR3 as shown in SEQ ID NO:

6.

2. The liquid preparation according to claim 1, characterized in that The VL of the anti-TNFR2 antibody comprises the amino acid sequence shown in SEQ ID NO:7, and the VH comprises the amino acid sequence shown in SEQ ID NO:8; Preferably, the anti-TNFR2 antibody comprises a light chain having an amino acid sequence as shown in SEQ ID NO:9, and a heavy chain having an amino acid sequence as shown in SEQ ID NO:10, and the anti-TNFR2 antibody is preferably a full-length antibody, Fab, Fab', F(ab')2, Fv such as scFv or di-scFv, VHH or a heavy chain antibody (HCAb); More preferably, the concentration of the anti-TNFR2 antibody is 5 to 200 mg / ml, preferably 10 to 100 mg / ml.

3. The liquid preparation according to claim 1 or 2, characterized in that The solvent is: (a) water, such as ultrapure water, preferably further comprising a surfactant; more preferably, the surfactant is a polysorbate surfactant, such as 0.1 to 2 mg / ml polysorbate 80; Or, (b) a buffer solution, wherein the pH of the buffer solution is 4.0 to 7.0; preferably, the buffer solution comprises one or more of an acetate buffer system, a phosphate buffer system, a histidine hydrochloride buffer system and a histidine acetate buffer system; more preferably, the concentration of the buffer solution is 5 to 50 mM, preferably 5 to 30 mM.

4. The liquid preparation according to any one of claims 1 to 3, characterized in that The liquid preparation further comprises: (iii) stabilizers; Preferably, the stabilizer comprises an osmotic pressure regulator and / or an antioxidant: the osmotic pressure regulator is one or more of sodium chloride, sucrose, trehalose, sorbitol, arginine, glycine and proline; and / or the antioxidant is one or more of glycine, methionine, arginine hydrochloride, sodium chloride and disodium edetate; More preferably, the concentration of the osmotic pressure regulator is 1 to 300 mM, preferably 100 to 300 mM, more preferably 147 to 273 mM, and the concentration of the antioxidant is 1 to 50 mM, preferably 1 to 20 mM, more preferably 7 to 13 mM.

5. The liquid preparation according to any one of claims 1 to 4, characterized in that The liquid preparation further comprises: (iv) surfactant; Preferably, the surfactant is poloxamer 188 or a polysorbate surfactant, such as polysorbate 80; More preferably, the liquid preparation further comprises: (v) a chelating agent, preferably edetate disodium; Further preferably, the concentration of the surfactant is 1-2 mg / ml, preferably 0.05-0.5 mg / ml, more preferably 0.1-0.15 mg / ml; the concentration of the chelating agent is 0.01-0.1 mg / mL, preferably 0.01-0.05 mg / mL, for example 0.019 mg / mL.

6. The liquid preparation according to any one of claims 1 to 5, characterized in that The liquid preparation is an injection, preferably for subcutaneous injection or intravenous injection, or an infusion, for example for intravenous infusion.

7. A solid antibody preparation obtained by solidifying the liquid preparation according to any one of claims 1 to 6, wherein the solid antibody preparation is in the form of a lyophilized powder injection.

8. A method for preparing a liquid preparation, characterized in that: The anti-TNFR2 antibody is added to a solvent preferably containing a stabilizer, a surfactant and / or a chelating agent to form the liquid preparation according to any one of claims 1 to 6.

9. A delivery device comprising the liquid preparation according to any one of claims 1 to 6 or the solid antibody preparation according to claim 7.

10. A prefilled syringe comprising the liquid preparation according to any one of claims 1 to 6 or the solid antibody preparation according to claim 7, for intravenous injection or intramuscular injection.

11. Use of the liquid preparation according to any one of claims 1 to 6 or the solid antibody preparation according to claim 7 in the preparation of diagnosis, prevention and / or treatment of cancer; Preferably, the cancer is ovarian cancer, acute myeloid leukemia, Lewis lung cancer, breast cancer, hepatocellular carcinoma, neural cancer and / or colorectal cancer.

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